Mycobacteria are gram-positive bacilli, nonmotile rod-shaped organisms that do not form spores. The composition of the cell wall includes a very high concentration of lipids complexed to a variety of peptides and polysaccharides. The unusual structure of the cell wall distinguishes mycobacteria from most other bacteria and is detectable by its resistance to acid-alcohol staining.
The disease caused by M. tuberculosis is a progressive, deadly illness that tends to develop slowly and follows a chronic course (Plorde, 1994). It is presently estimated that one-third of the world""s population is infected with M. tuberculosis, 30 million of whom have active disease (Plorde, 1994). An additional 8 million people develop the disease annually (Plorde, 1994). Most infections are caused by inhalation of droplet nuclei carrying the mycobacterium. A single cough can generate 3000 infected droplet nuclei and even 10 bacilli may be sufficient to cause a pulmonary infection. In addition to the primary infection, reactivation of the disease can occur in older people and in immunocompromised patients.
When intracellular pathogens, such as Mycobacterium tuberculosis, are ingested by macrophages the bacteria are under environmental stress. The genes required for survival following uptake by macrophages can provide insight into mycobacterial pathogenesis, and provide novel targets for developing antibacterial agents. The ability to adapt to the intracellular stress requires regulation of complex gene expression and this regulation may be mediated in part by one or more alternative sigma factors. Therefore stress response alternative sigma factors (sigE family) from M. tuberculosis are potential novel targets for antibacterial therapeutics.
Extracellular environmental stress can significantly affect the survival of the bacteria. As part of the adaptive response by the bacteria the alternative sigma factors play a critical role in coordinate regulation of gene expression. For example, survival following extreme temperature in Escherichia coli is regulated by a family of alternative sigma factors known as the sigE family (Keiichiro et al., Raina et al., Rouviere et al.). Alginate production in Pseudomonus aeruginosa is also regulated by the sigE family member known as the algU gene (Deretic et al.). Respiratory infections with mucoid P. aeruginosa in cystic fibrosis (CF) patients are the major cause of mortality. Although initial colonizing strains are nonmucoid, the bacteria are converted to mucoid P. aeruginosa in the CF lung. This conversion to mucoidy is regulated by the alternative sigma factor algU (Martin et al.).
Sigma ("sgr") factors are positive regulators of general transcription initiation that enhance transcriptional specificity. The basic unit of the eubacterial transcription apparatus is the DNA-dependent RNA polymerase holoenzyme, a complex consisting of five protein subunits: two copies of the xcex1 subunit and one copy each of the xcex2, xcex2xe2x80x2, and "sgr" subunits. The xcex1, xcex2 and xcex2xe2x80x2 subunits are invariant in a given bacterial species and together form core RNA polymerase. Open promoter complexes form only when holoenzyme is bound at a promoter (Gross et al., 1992). When the newly synthesized RNA chain is 8-9 nucleotides long, "sgr" factor dissociates from the complex and the elongation process is begun (von Hippel, et al., 1992). After transcription is terminated, "sgr" factor rebinds core polymerase, creating holoenzyme for another round of initiation (von Hippel, et al., 1992). This series of biochemical activities has been termed xe2x80x9cthe transcription cyclexe2x80x9d.
Rifampicin, a highly specific inhibitor of mycobacterium/RNA polymerase, is one of the primary drugs of choice for treatment of tuberculosis. Combination treatment with isoniazid is typical if there is no risk of developing multi-drug resistance. Prolonged treatment regimens are necessary and can take up to nine months. Failure to complete the prolonged treatment course is one of the contributing factors in the development of resistant bacterial strains. Rifabutin is an effective analog of rifampicin, but 70% of rifampicin-resistant strains are also rifabutin-resistant.
Although RNA polymerase is a well-validated target for anti-mycobacterial therapy, discovery of inhibitors of M. tuberculosis RNA polymerase is hampered by a lack of information concerning components of the M. tuberculosis transcriptional apparatus, difficulties in obtaining sufficient yields of active enzymes for biochemical studies, and technical and biosafety concerns surrounding the handling of live cultures of M. tuberculosis. Establishment of an in vitro transcription system employing purified and reconstituted RNA polymerase would greatly advance efforts to identify new therapeutic agents active against tuberculosis. It is very possible that molecules that inhibit a functions may not affect eukaryotic general transcription. Thus, "sgr" factors are a reasonable target for development of transcriptional inhibitors. Therefore, molecules that inhibit "sgr" factor function may be used as general transcriptional inhibitors and antibacterial therapeutics.
Accordingly, there is a need in the art for compositions and methods utilizing cloned genes and purified proteins derived from M. tuberculosis RNA polymerase.
The present invention is based on the isolation and characterization of DNA encoding the "sgr" subunit of RNA polymerase derived from the algU gene from M. tuberculosis. In one aspect, the invention provides a purified, isolated nucleic acid having the sequence shown in FIG. 3 SEQ ID NO:1. The invention also encompasses sequence-conservative and function-conservative variants of this sequence. The invention also provides vectors comprising these sequences, and cells comprising the vectors.
In another aspect, the present invention provides a purified, isolated polypeptide encoded by the nucleic acid sequence shown in FIG. 3, as well as function-conservative variants thereof.
In yet another aspect, the invention provides in vitro methods for high-throughput screening to detect inhibitors of M. tuberculosis RNA polymerase. The methods are carried out by the steps of:
a) providing a mixture comprising
(i) purified M. tuberculosis RNA polymerase containing the algU "sgr" factor and
(ii) a DNA template encoding a promoter sequence that is recognized by M. tuberculosis RNA polymerase containing the algU subunit;
b) incubating the mixture in the presence of test compounds to form test samples, and in the absence of test compounds to form control samples, under conditions that result in RNA synthesis in the control samples;
c) measuring RNA synthesis in the test and control samples; and
d) comparing the RNA synthesis detected in step (c) between the test and control samples. According to the invention, an RNA polymerase inhibitor is a test compound that causes a reduction in RNA synthesis measured in the test sample relative to RNA synthesis measured in the control sample.
In yet another aspect, the invention provides in vivo methods for high-throughput screening to detect inhibitors of M. tuberculosis RNA polymerase. The methods are carried out by the steps of:
a) providing a non-mycobacterial bacterial strain, preferably E. coli, that
(i) has been transformed with a DNA template encoding a promoter sequence that is recognized by M. tuberculosis RNA polymerase containing the algU subunit, and
(ii) expresses enzymatically active M. tuberculosis RNA polymerase (e.g., xcex1, xcex2, xcex2xe2x80x2 plus the algU "sgr" subunit disclosed herein);
b) incubating the bacterial strain of (a) in the presence of test compounds to form test samples, and in the absence of test compounds to form control samples;
c) measuring RNA synthesis in the test and control samples; and
d) comparing the RNA synthesis detected in step (c) between the test and control samples. According to the invention, an RNA polymerase inhibitor is a test compound that causes a reduction in RNA synthesis measured in the test sample relative to RNA synthesis measured in the control sample.
These and other aspects of the present invention will be apparent to those of ordinary skill in the art in light of the present specification and appended claims.